Functional Characterization and Potential Applications for Enhanced Green Fluorescent Protein- and Epitope-Fused Human M1 Muscarinic Receptors Claire Weill, *Jean-Luc Galzi, ²Sylvette Chasserot-Golaz, Maurice Goeldner, and Brigitte Ilien Laboratoire de Chimie Bio-Organique, UMR 7514 CNRS; and *De ´partement Re ´cepteurs et Prote ´ines Membranaires, UPR 9050 CNRS, Illkirch; and ² Unite ´ Biologie de la Communication Cellulaire, U. 338 INSERM, Strasbourg, France Abstract: Four recombinant human M1 (hM1) muscarinic acetylcholine receptors (mAChRs) combining several modifications were designed and overexpressed in HEK293 cells. Three different fluorescent chimera were obtained through fusion of the receptor N terminus with enhanced green fluorescent protein (EGFP), potential gly- cosylation sites and a large part of the third intracellular (i3) loop were deleted, a hexahistidine tag sequence was introduced at the receptor C terminus, and, finally, a FLAG epitope was either fused at the receptor N terminus or inserted into its shortened i3 loop. High expression levels and ligand binding properties similar to those of the wild-type hM1 receptor together with confocal micros- copy imaging demonstrated that the recombinant pro- teins were correctly folded and targeted to the plasma membrane, provided that a signal peptide was added to the N-terminal domain of the fusion proteins. Their func- tional properties were examined through McN-A-343- evoked Ca 2+ release. Despite the numerous modifica- tions introduced within the hM1 sequence, all receptors retained nearly normal abilities (EC 50 values) to mediate the Ca 2+ response, although reduced amplitudes (E max values) were obtained for the i3-shortened constructs. Owing to the bright intrinsic fluorescence of the EGFP- fused receptors, their detection, quantitation, and visual- ization as well as the selection of cells with highest ex- pression were straightforward. Moreover, the presence of the different epitopes was confirmed by immunocyto- chemistry. Altogether, this work demonstrates that these EGFP- and epitope-fused hM1 receptors are valuable tools for further functional, biochemical, and structural studies of muscarinic receptors. Key Words: Muscarinic receptor—Human M1 subtype—Fluorescent chimera— Enhanced green fluorescent protein—Histidine tag— FLAG epitope. J. Neurochem. 73, 791– 801 (1999). Muscarinic acetylcholine receptors (mAChRs), of which five subtypes have been identified and termed M1–M5, are members of the G protein-coupled receptor (GPCR) superfamily (Ashkenazi and Peralta, 1994). Structural descriptions of their ligand binding domains were achieved mainly through site-directed mutagenesis (Wess, 1993, 1997), site-directed affinity labeling (Spal- ding et al., 1994), and molecular modeling (Bourdon et al., 1997). We previously reported on the efficient alkylating properties of two photoactivatable aryldiazonium deriv- atives at both membrane-bound (Ilien and Hirth, 1989) and purified (Autelitano et al., 1997; Weill et al., 1997) mAChRs. As the same probes brought considerable structural information on the acetylcholine binding site of the nicotinic receptor (Dennis et al., 1988; Galzi et al., 1990) and on the active site of cholinesterases (Harel et al., 1993; Nachon et al., 1998) in labeling several key amino acid residues, we consider them as promising tools to investigate the muscarinic ligand binding site. On a practical point of view, biochemical analyses of mAChRs are hindered by several factors, such as their paucity and their subtype diversity in natural sources, their molecular heterogeneity due to glycosylation and proteolytic processes, and finally the transmembrane lo- cation of their ligand binding site. We decided to over- come several of these problems by engineering a well- defined mAChR subtype, the human M1 (hM1) receptor, and introducing appropriate and nondeleterious modifi- cations. In a previous communication (Weill et al., 1999), we reported on the successful establishment of one chimeric fluorescent hM1 receptor (Chim A2) and its preliminary characterization. Received January 8, 1999; revised manuscript received March 4, 1999; accepted March 11, 1999. Address correspondence and reprint requests to Dr. B. Ilien at Laboratoire de Chimie Bio-Organique, UMR 7514 du CNRS, Faculte ´ de Pharmacie, 74, route du Rhin, B.P. 24, 67401 Illkirch cedex, France. Abbreviations used: EGFP, enhanced green fluorescent protein; FACS, fluorescence-activated cell sorting; p-f-HHSiD, p-fluorohexa- hydrosiladifenidol; GPCR, G protein-coupled receptor; HEK293, hu- man embryonic kidney 293; hM1, human M1 muscarinic receptor subtype; i3, third intracellular; mAChR, muscarinic acetylcholine re- ceptor; McN-A-343, 4-[N-(3-chlorophenyl)carbamoyloxy]-2-butynyl- trimethylammonium chloride; [ 3 H]NMS, [N-methyl- 3 H]scopolamine methyl chloride; [ 3 H]QNB, [ 3 H]quinuclidinyl benzilate; wt, wild-type. 791 Journal of Neurochemistry Lippincott Williams & Wilkins, Inc., Philadelphia © 1999 International Society for Neurochemistry